Personal care composition containing yeast/polyphenol ferment extract

ABSTRACT

A method of producing yeast-polyphenol ferment extract includes growing the cells of a yeast of  pichia  genus in a growth media to late-logarithmic growth phase, in which the carbon source in the media is exhausted; exposing the cells to a non-cytotoxic dose of polyphenol; and extracting cytoplasmic and extra-cellular components of the exposed yeast cells from the growth media.

CROSS REFERENCES TO RELATED APPLICATIONS

This is a continuation application of application Ser. No. 12/460,725,filed on Jul. 23, 2009, which claims the priorities and benefits of U.S.Provisional Patent Application Ser. No. 61/135,992, filed on Jul. 24,2008, the disclosures of which are incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates generally to personal care compositions,and methods of making the same. More specifically, the invention relatesto personal care compositions containing a yeast-polyphenol fermentextract.

BACKGROUND OF THE INVENTION

Recently, the use of yeast and its derivatives has become very popularin cosmetic applications. This is driven by the fact that yeast, beingeukaryotic, has similar cellular biological processes to human cells andis known to trigger the production of beneficial proteins under stress.There is also considerable historical evidence for the use of yeastderivatives and their benefits in topical skin applications by theincrease in oxygen uptake. For example, U.S. Pat. No. 4,540,571discloses yeast derivatives that improve cutaneous respiration. Yeastcell derivatives are also reported to stimulate the production ofcollagen and elastin in skin cells.

Recent studies suggest that yeast cell extracts have the ability topromote ‘growth factors’ that stimulates wound healing. For example,U.S. Pat. No. 7,217,417 discloses a method that incorporates yeast cellderivatives into a gel-based formulation for wound healing.

For topical applications, the most commonly used yeast is Saccharomycescerevisiae, also known as baker's yeast. Saccharomyces is categorized bythe FDA as a “Generally Recognized as Safe (GRAS)” micro-organism.However, products containing Saccharomyces cerevisiae or its derivatesnormally have strong color and characteristic odor. Therefore, there isa need in the cosmetic industry for yeast and yeast extracts thatprovide beneficial effects to skin's, as well as desirable aestheticvalues at the same time.

Resveratrol (3, 4, 5-transhydroxystilbene), a polyphenol found inseveral plants, has been of considerable interest due to its possiblehealth benefits for treatment of cardiovascular disease, diabetes andcancer. Resveratrol is well known for its presence in red wine and canbe extracted from grape skin. Resveratrol can also be found in goodabundance in the leaves of various plants such as, for example, JapaneseKnotweed (Polygonum cuspidatum). Resveratrol is considered to be relatedto a family of biologically active molecules isolated from plants thatare routinely called or referred to as polyphenols. Polyphenols are agroup of chemical substances found in plants, characterized by thepresence of more than one phenol unit or building block per molecule.Polyphenols include, for example, flavonoids or isoflavonoids,catechins, anthrocyanins and proanthrocyanins.

Several studies in wide range of species have demonstrated the abilityof resveratrol to positively affect cellular function and longevity. Itis suggested that resveratrol affects cellular longevity by mimickingcaloric restriction without requiring any reduction in calorie intake.This is accomplished by the activation of specific proteins, mainlybelonging to the “Sirtuins” family of proteins as disclosed by Borra T.M. et. al. in J. Biol Chem. 280, (2005) 17187.

In addition to caloric restriction, resveratrol is also being evaluatedfor its potential health benefits including triggering DNA-repairmechanisms, anti-carcinogenic and anti-oxidant properties.

Ciolino H. P. et al. discloses that resveratrol may be used to reversethe aging process due to cellular DNA damage. Mol Pharm 56 (1999) 4,760. In addition, resveratrol has been found to inhibit the activity ofseveral inflammatory enzymes in vitro, including cyclooxygenases andlipoxygenases. Pinto M. D., et al. “Resveratrol is a potent inhibitor ofthe dioxygenase activity of lipoxygenase.” J Agic Food Chem. 47(12),4842-4846 (1999).

Resveratrol has demonstrated advantages in topical and oralapplications, but it suffers from inherent chemical instability and hasbeen shown to be somewhat cytotoxic to skin cells in its native form. Itis also quite water-insoluble which makes formulating skin and hair careproducts with the molecule difficult. A need remains to develop novelingredients based on resveratrol that are more beneficial and lessdifficult to work with. As such, it is appreciated that there is a needfor an ingredient that provides combined advantages effects ofresveratrol and the metabolic pathways of yeast, for use in personalcare compositions.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a yeast-polyphenol fermentextract produced by a method comprising the steps of: growing the cellsof a yeast of Pichia genus in a media to late-logarithmic growth phasewherein the carbon source in the media is exhausted; exposing the cellsto a non-cytoxic dose of polyphenol as a growth factor to generate newactive ingredients through metabolic pathways of the yeast; andseparating the new active ingredients by filtration or lysing to providethe yeast-polyphenol ferment extract containing the new actives. In apreferred embodiment, the polyphenol is resveratrol dispersed in anorganic solvent at a concentration of from about 0.001 gram/liter to 1.0gram/liter; and the yeast is Pichia pastoris.

Another aspect of the invention relates to a personal care compositioncomprising a yeast-polyphenol ferment extract and a preservative,wherein the preservative is selected from the group consisting of acids,alcohols, glycols, parabens, quaternary-nitrogen containing compounds,isothiazolinones, aldehyde-releasing compounds, halogenated compoundsand combinations thereof. The yeast-polyphenol ferment extract ispresent in a concentration between 0.0001% and 95%, preferably0.01%-50%, most preferably 0.01%-10% by weight based on the total weightof the personal care composition. The preservative is present in aconcentration between 0.01%-10% by weight based on the total weight ofthe personal care composition. The personal care composition may furthercomprise a cosmetically acceptable vehicle, and other activeingredients.

A further aspect of the present invention relates to a method ofpreparing a personal care composition including providing an effectiveamount of yeast-polyphenol ferment extract and incorporate the extractinto a personal care composition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting the changes of fermentation parameters overtime during a yeast/resveratrol fermentation process.

FIG. 2 shows a 2D gel electrophoresis comparison of a non-treated yeastextract (control) and a resveratrol treated yeast extract.

FIG. 3 shows a 2D gel laser densitometer comparison of a non-treatedyeast extract (control) and a resveratrol treated yeast extract.

FIG. 4 is a graph of relative expression of genes by the treatment of ayeast/resveratrol ferment extract on human epidermal keratinocytes.

FIG. 5 is a graph illustrating the effect of yeast/resveratrol fermentextracts to COX1 enzyme.

FIG. 6 is a graph illustrating the effect of yeast/resveratrol fermentextracts to Type IV collagen protein.

FIG. 7 is a graph illustrating the effect of yeast/resveratrol fermentextracts to the expression of melanin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The ‘yeast/polyphenol ferment extract’ of the present invention isobtained through a specialized process called fermentation. Fermentationoccurs when living eukaryotic and/or prokaryotic microorganisms aregrown on a nutrient media either in the presence of oxygen (known asaerobic fermentation) or the absence or diminishment of oxygen (known asanaerobic fermentation). The fermentation process can take place withone microorganism, or simultaneously or sequentially with two or moremicroorganisms (often referred to as co-ferments). The nutrient media istypically a well-defined mixture of proteins, sugars, minerals and thelike. Such media is known to a person skilled in the art and may beavailable through a variety of commercial sources.

The process of metabolizing yeast can occur with a variety ofmicroorganisms such as, for example, yeast, bacillus, molds, plant cellsand the like. Especially preferred for the composition of the presentinvention are ferments made using yeast. As used herein, the term“yeast” is meant to encompass a single yeast cell, multiple yeast cellsand/or a culture of yeast cells. The yeast of the present invention canbe of various fungal families, known to those skilled in the artincluding, but not limited to: Neurospora, Ceratostomella, Claviceps,Xylaria, Rosellinia, Helotium, Sclerotinia, Tulostoma, Rhizopogon,Truncocolumella, Mucor, Rhizopus, Entomophthora, Dictostylium,Blastocladia, Synchytrium, Saprolegnia, Peronospora, Albugo, Pythium,Phytophthora, Plasmodiophora, Tuber, Hydnum, Lecanora, Roccella,Pertusaria, Usnea, Evernia, Ramalina, Alectoria, Cladonia, Parmelia,Cetraria, Agaricus, Cantharellus, Omphalotus, Coprinus, Lactarius,Marasmius, Pleurotus, Pholiota, Russula, Lactarius, Stropharia,Entoloma, Lepiotaceae, Corticium, Pellicularia, Tricholoma, Volvaria,Clitocybe, Flarnmulina, Saccharomyces, Schizosaccharomyces,Saccharomycetaceae, Eurotium, Aspergillus, Thielavia, Peziza, Plectania,Morchella, Wynnea, Helvella, Gyromitra, Phallales, Dictyophera, Mutinus,Clathrus, Pseudocolus, Lycoperdon, Calvatia, Geastrum, Radiigera,Astreus, Nidularia, Gastrocybe, Macowanites, Gastroboletus, Albatrellus,Neolentinus, Nigroporus, Oligoporus, Polyporus, Fistulina, Fomes,Boletus, Fuscoboletinus, Leccinum, Phylloporus, Suillus, Strobilomyces,Boletellus, Tremella, Auricularia, Dacrymyces, Melampsora, Cronartium,Puccinia, Gymnosporangium, Tilletia, Urocystis, Septobasidium,Hygrocybe, Hygrophorus, Hygrotrama, Neohygrophorus, Cortinarius,Gymnopilus, Trichophyton, Microsporum, Monilia, Candida, Cercosporella,Penicillium, Blastomyces, Cercospora, Ustilaginoidea, Tubercularia,Fusarium, Rhizoctinia, Ozonium, Sclerotium, Geoglossum, or Armillaria.Of particular interest are the fungi belonging to the familySaccharomycetaceae. Of greater interest are the fungi belonging to thegenus Pichia. Of most interest are the fungi belonging to the speciespastoris. In a preferred embodiment, Pichia pastoris is used in thefermentation process.

Typically, yeasts belonging to the phylum Ascomycetes are facultativeanaerobes; that is they can grow both in the presence or absence ofoxygen. For the purpose of the present invention, yeast was aerobicallygrown and therefore air is critical for growth.

Polyphenols are significant components found in plants and fruits thatare responsible for a variety of well known benefits for skin (SvobodováA , Jitka Psotová, Daniela Walterová. Natural phenolics in theprevention of UV-Induced skin damage. Biomed. Papers 147(2), 137-1452003). Generally polyphenols are extracted from plants. For purposes ofthis invention, polyphenols can also be prepared by synthetic means.Preferably, the polyphenols suitable for use in the fermentation processof the present invention has a purity of greater than 50%, morepreferably, greater than 70% and most preferably, greater than 80%.

It has been surprisingly found that when a yeast belonging to the genusof Pichia, for example, Pichia pastoris, is exposed to a non-cytotoxicamount of a polyphenol, for example, resveratrol, as a growth factorduring a key stage of fermentation, the yeast changes its metabolicpathways and produces new actives that are beneficial to the skin. Theproduced new actives are relatively stable and do not undergo rapidconversions into less-desired metabolites. Meanwhile, the new activesalso have desirable color and odor thus making them an improvedingredient for personal care compositions.

The yeast/polyphenol extracts of the present invention includecytoplasmic and extra-cellular components of the yeast which include,but are not limited to, the nutrient broth, cellular protein material,cellular nucleic material, cellular protoplasmic material and/or cellwall components. Typically, the extracts are relatively water soluble,for example, equal or more than 1-gram of yeast extracts dissolve in1-gram of water. The extracts may also be soluble in water/organicsolvent mixtures such as, but not limited to, aqueous glycols andaqueous glycerols.

The yeast fermentation process can be carried out in a stirred tankbio-reactor. Examples of such bioreactors might include for example,fermentors available from New Brunswick Scientific, Edison N.J. orApplikon Biotechnology Foster City Calif.

To produce a ‘yeast/polyphenol ferment extract’ of the presentinvention, Pichia pastoris cell culture can be grown between 25° C. to32° C. in a growth media. An example of a typical yeast fermentationmedia can be typically found in the “Handbook of Microbiological Media”published by CRC press. For the purpose of this invention, a specificgrowth media for fermentation of yeast is used. This growth media ischemically defined media and does not have any animal-derived products.In a preferred embodiment, the sole source of carbon in the media ofpresent invention is from glycerol.

To initiate the polyphenol feeding strategy, a non-cytotoxic dose ofpolyphenol can be dispersed in an organic solvent that is not toxic tothe cells to provide a polyphenol feed batch. As used herein,non-cytotoxic dose is defined as a dose where there is no loss in cellviability. In a preferred embodiment, the concentration of thepolyphenol in the organic solvent is at least from 0.001 gram/liter to1.0 gram/liter.

According to one embodiment of the invention, the polyphenol feed-batchis initiated when the cells are in their late-logarithmic growth phaseand the carbon source in the media is exhausted (as confirmed bymonitoring carbon source in the bioreactor). For polyphenols that arehighly hydrophobic, it is critical to monitor the fermentation processto ensure that these polyphenols do not precipitate in the aqueousgrowth media.

The ‘yeast/polyphenol ferment extract’ can be obtained by firstseparating the bio-mass and then extracting active ingredients from theextra-cellular secretions of the yeast. Alternatively, the yeast cellcan be lysed to obtain ‘yeast/polyphenol ferment lysate’ by processesknown to those skilled in the art. Typically, the process involves therupture of the yeast cell walls by chemical, enzymatic or physical meansor by combination of these. The ‘yeast/polyphenol ferment extract’ maybe further purified by chromatography, solvent extraction,centrifugation, decantation, filtration or carbon treatment, or othermeans known to a person skilled in the field. In one embodiment, the‘yeast/polyphenol ferment extract’ is further concentrated by any meansknown to those, skilled in the art, for example, evaporation,spray-drying, lyophylization, belt or drum drying.

After exposing the yeast, for example, Pichia pastoris, to polyphenolgrowth factor during a key phase of the fermentation process, the yeasthas significant genomic and proteomic changes compared to the yeast thatis not subjected to the treatment of the polyphenol. In addition,examination of yeast microarrays indicates that various genes within thefermenting pichia pastoris are either upregulated or downregulated as aresult of treatment with the polyphenol. The terms upregulated anddownregulated refer to the influence the polyphenol has on signallygenes within the yeast to either be turned-on (upregulated) orturned-off (downregulated). The up and down-regulation of the yeastgenes can result in changes in protein expression which allows the yeastto express proteins that would not normally be expressed if thepolyphenol were not present.

In addition, the Pichia pastoris may ingest the polyphenol and begin toconvert the polyphenol into chemical derivatives such as, for example,sugar or protein derived metabolites of polyphenol. The yeast might dothis as a means to begin the process of digesting the polyphenol. Inparticular, if the yeast chemically attaches a protein to the polyphenolon one of the available hydroxyl groups on polyphenol, such moleculesmight appear in 2D-gel electrophoresis as new proteins. It is difficultto differentiate these new chemically altered polyphenol molecules fromnormal metabolic proteins that the yeast typically expresses. However,some techniques such as MALDI-TOF Mass Spectroscopy can be used toidentify proteins from 2D-gels. In addition, one can examine protein andsugar metabolites of polyphenol by high performance liquidchromatography (HPLC). Without being bound by theory, it is believedthat the secondary metabolites of polyphenol formed by fermentation mayplay a significant role in the ability of the yeast/polyphenol fermentextract to stimulate human skin in beneficial ways not available to justthe yeast or the polyphenol alone.

The effect of the yeast/polyphenol ferment extract to influence humanskin can be measured in a number of ways known to those skilled in theart. In particular, the yeast/polyphenol ferment extracts can bescreened for their effects on skin by employing analytical techniquessuch as, for example, human genomic microarrays on specific skin cellssuch as keratinocytes or fibroblasts, or by protein expression analysison individual skin cells, tissue models or ex vivo or in vivo skinmodels. In these testing models, specific genes and or proteins may beup-regulated or down-regulated as a result of the extract treatment.Genes and proteins that are capable of regulating skin conditions are ofparticular interest in the screening. Of particular interest for thepurpose of the present invention are genes and proteins related toinflammation, extracellular matrix expression, melanin regulation, skinmoisturization, exfoliation and the like. Of particular interest areproteins related to cyclooxygenase expression, in particularcyclooxygenase-1 and 2 and also extracellular matrix protein expression,in particular, types, I, IV and VI collagen expression. In addition, theinfluence of the extract on skin melanin expression is also ofconsiderable interest.

The effect of the yeast/polyphenol ferment extract on expression ofthese and other important cutaneous proteins can be monitored by humangenomic microarray analysis and protein expression as well as bynon-invasive test methods well-known to those in skilled in the art,including, but not limited to, improved moisturization, wrinklereduction, reduced pigmentation, improved skin tone and the like.Application of the yeast/polyphenol ferment extract can manifest itselfby measured reductions in skin wrinkles, for example, as measured bytechniques such as SilFlo silicone modeling, PRIMOS and VISIOphotographic systems and the like. In addition, moisturization might bemeasured using transepidermal water loss (TEWL) or cutometer orcomeometric measurements. Likewise, skin pigmentation could be measuredusing a chromometer. Such testing technologies are well-known to thoseskilled in the art and can be found in Handbook of non-invasive methodsand the skin, 2^(nd) edition, Serup J, Jemec GBE, Grove GL (ed.), Taylorand Francis Boca Raton Fla. 2006, the disclosure of which isincorporated herein by reference in its entirety.

As shown, for example, in FIG. 4, human gene microarray analysis onhuman epidermal keratinocytes indicates that the yeast-resveratrolferment extract up-regulates several genes involved in various skinmetabolism functions. It is thus appreciated that the yeast-polyphenolferment extract according to the present invention can be used inpersonal care compositions.

The personal care composition of the present invention may contain aneffective amount of ‘yeast/polyphenol ferment extract’ and at least onepreservative selected from the group consisting of acids, alcohols,glycols, parabens, quaternary-nitrogen containing compounds,isothiazolinones, aldehyde-releasing compounds and halogenatedcompounds. Illustrative alcohols include phenoxyethanol, isopropylalcohol, and benzyl alcohol; illustrative glycols include propylene,butylene and pentylene glycols; illustrative parabens include (alsoknown as parahydroxybenzioc acids) methyl, propyl and butyl parabens;illustrative quaternary nitrogen containing compounds includebenzalkonium chloride, Quartenium 15; illustrative isothiazoles includemethylisothiazoline, methychlorolisothiazoline; illustrative aldehydereleasing agents include DMDM hydantion, imiadolidinyl urea anddiazolidinyl urea; illustrative anti-oxidants include butylatedhydroxytoluene, tocopherol and illustrative halogenated compoundsinclude triclosan and chlorohexidene digluconate. Examples ofpreservatives useful for the purpose of the present invention can befound in Steinberg, D. “Frequency of Use of Preservatives 2007”. Cosmet.Toilet. 117, 41-44 (2002) and, “Preservative Encyclopedia” Cosmet.Toilet. 117, 80-96 (2002). In addition, enzyme preservative systems suchas those described in the article by Ciccognani D. Cosmetic PreservationUsing Enzymes. in “Cosmetic and Drug Microbiology”, Orth DS ed., Francis& Taylor, Boca Raton, Fla. (2006) can also be effective for use with thecomposition of the present invention.

The amount of ‘yeast/polyphenol ferment extract’ present in a personalcare composition is between 0.001% and 95% based on the total weight ofthe personal care composition, preferably between 0.01% and 50%, morepreferably between 0.01% and 10%. The preservative is present in aconcentration between 0.01% and 10% by weight based on the total weightof the composition.

Additionally the ‘yeast/polyphenol ferment extract’ can optionallycontain other functional ingredients such as, water, surfactants,emulsifiers, conditioners, emollients, waxes, oils, polymers,thickeners, fixatives, colorants, humectants, moisturizers, stabilizers,diluents, solvents and fragrances. In addition the personal carecomposition may contain active ingredients such as botanicals,nutraceuticals, cosmeceuticals, therapeutics, pharmaceuticals,antifungals, antimicrobials, steroidal hormones, antidandruff agents,anti-acne components, sunscreens, preservatives and the like.

The ‘yeast/polyphenol ferment extract’ can be used in various types ofcosmetic formulations including, but not limited to lotions, ointments,creams, sprays, spritzes, aqueous or aqueous-alcoholic mixture gels,mousses, patches, pads, masks, moistened clothes, wipes, solid sticks,clear sticks, lip sticks, aerosol creams, anhydrous powders, talcs,tonics, oils, emulsions and bath salts. Such cosmetic formulations maybe used as a topical application on the skin.

The ‘yeast/polyphenol ferment extract’ of the present invention manyinclude the extract alone or they may include the ‘yeast/polyphenolferment extract’ encapsulated in various chemical delivery vehiclesincluding, but not limited to, liposomes, niosomes, sub-micronemulsions, polymeric encapsulates, gels, creams, lotions, andcombinations thereof.

The following examples are intended to illustrate the art of the presentinvention and are not intended to limit the scope of the claims below.

EXAMPLE—1 Preparation of Yeast/Reveratrol Ferment Extract

Organism and Media

Yeast cell culture was obtained from ATCC (Pichia pastoris # 60372).Stock cultures were maintained on yeast-peptone-dextrose (YPD) agarplates. The parent stock culture was grown in YPD broth and maintainedat −20° C. The fermentation was carried out in Yeast Nitrogen-Base (YNB)growth media and supplemented with glycerol containing, 2.7% H3PO4,0.09% CaSO₄, 1.8% K₂SO₄, 1.5% MgSO₄, 0.41% KOH, 4% glycerol (Sigma St.Louis, Mo.). Resveratrol (>98%) was purchased from Wilshire Technologies(Princeton. N.J.). Antifoam sigma-emulsion B was used throughout theprocess (Sigma, St. Louis, Mo.).

Fermentor

After optimization of treatment via the shake flask trials, the processwas scaled up to 2 L and 15 L fermentation stages (2 L New BrunswickScientific, Edison N.J. and 15 L Applikon Biotechnology Foster CityCalif.).

Stress Conditions

Fed-batch cultures were grown at 29° C., in Yeast Nitrogen-Base (YNB)growth media and supplemented with glycerol (2.7% H₃PO₄, 0.09% CaSO₄,1.8% K₂SO₄, 1.5% MgSO₄, 0.41% KOH, 4% glycerol). The pH was keptconstant at 5.0±0.5 by titration with 2M NH₄OH. The dissolved oxygenlevels were measured by a sterilizable DO probe and the oxygensaturation was kept at 30% by regulating the stirring velocity between100 and 600 rpm. The aeration rate was set at 1 VVM. Resveratrol feedwas initiated at the concentration of 0.001 mg/ml. This method wassuccessfully scaled up from bench-top to pilot-scale to production.

An overview of the yeast/resveratrol fed-batch fermentation process isshown in FIG. 1. The pH, temperature and other fermentation parameterswere constantly monitored to reflect the batch process. Theresveratrol-feed stage focuses on uptake of resveratrol by themicroorganism, in a nutrient-limited environment.

For the purpose of further testing, samples collected at 4, 8, 12, 24hours after resveratrol feed was initiated, indicate that there is noevidence of toxicity of cells as measured by changes in optical densityand viable cell counts.

EXAMPLE—2 Effect of Yeast-Resveratrol Fermentation Process on YeastProtein Expression

2Dimensional Gel Electrophoresis

To identify the metabolic changes in the yeast because of the polyphenolfeed-strategy, 2-Dimensional gel electrophoreses and gene microarrayanalysis was conducted on the control, (i.e., a yeast extract withoutresveratrol growth factor), and a yeast-resveratrol ferment extractproduced by the process of the present invention. The first dimensionIEF focuses on separation of the proteins based on their isoelectricpoints and was conducted using the Ettan1PGphor system with a manifoldfor protein loading. The 2nd dimension SDS-PAGE is an electrophoreticmethod for separating polypeptides according to their molecular weightsusing the DALT VI Electrophoresis Unit (GE-Amersham, Piscataway N.J.)(2, 6). To remove interfering substances and impurities, the yeast cellextracts were purified using the 2D Clean-up kit provided by GEAmersham. Each purified yeast sample was rehydrated overnight withThiourea rehydration solution (7M Urea and 2M Thiourea, 2% Chaps, 0.5%Pharmalyte). The rehydrated samples were loaded onto the 1^(st)dimension IEF. Following the 1^(st) dimension IEF, the strips wereequilibrated and loaded onto the 2^(nd) dimension DALT VI. The gels werestained using Coomassie with sensitivity ranging from 10-500 ng proteinconcentration. Details of the 2Dimentional Gel Electrophoresis methodcan be found in United States Patent Application Publication No.US2006/0110815 A1, the disclosure of which is incorporated herein byreference in its entirety. The results of 2-Dimensional GelElectrophoresis for the control, i.e., the untreated yeast fermentextract, and the yeast-resveratrol ferment extract are shown in FIG. 2.

FIG. 3 illustrates the laser densitometer comparison of the controlversus the yeast/resveratrol of the invention. Reference spot numbering,pI, and MW are given for changing polypeptide spots and analyzed in thesamples. Polypeptide spots that are increased in yeast/resveratrol ofthe invention versus the control by a fold of >1.7 and have a p value<0.05 are highlighted in blue. A total of 626 spots were analyzed.

The results indicate that after exposing the yeast to resveratrol growthfactor during a key phase of the fermentation process, the yeast hassignificant proteomic changes (i.e., new proteins have either beenexpressed or older existing proteins are now being suppressed as aresult of the resveratrol treatment).

EXAMPLE—3 Gene Microarray Analysis on Human Epidermal Keratinocytes

After treatment with resveratrol in a procedure as illustrated inexample 1, the yeast cells were centrifuged and the pellet wasre-suspended in RNA Later (Ambion) and stored at 4° C. Total RNA wasextracted from the yeast using a RiboPure RNA extraction kit (Ambion),and mRNA was subsequently amplified using a Message AMP aRNA Kit(Ambion). For the array, samples of aRNA were fluorescently labeled witheither Cy-3 or Cy-5 using a MicroMax array labeling kit (Perkin Elmer).Labeled aRNA was then applied to a Yeast Genome DNA Microarray chip(Agilent Technologies), and hybridized overnight. On the following daythe chips were washed, scanned with an Axon 4100A DNA microarray scannerand analyzed using Axon Genepix-Pro® software. The method employed forgene microarray analysis can be found in United States PatentApplication Publication No. US2006/0110815.

The genes of interest obtained from the human epidermal keratinocytegene-microarray were carefully screened for their potential role in skinfunctions. The molecular chip generated from human epidermalkeratinocytes gene-microarray showed distinctive results in variousdifferentially expressed genes. Genes that were of interest wereobserved at different time periods of treatment to obtain atime-response for a specific gene. The gene expression was monitored toobtain a time-related response to the resveratrol nutritional strategy.The up-regulation of several genes involved in various skin metabolismfunctions were detected. As shown in FIG. 4, yeast/resveratrol fermentextract causes statistically significant changes in genes such as NOS1,FER1, DEFB4, LAMB3, COX1, COX2, COL4A1, COL1A1, COL1A2, COL6A1, COL6A2and SOD1. Numerous other genes such as NOS3, TMMP1, DHCR7 and VEGFA werealso up-regulated by the application of yeast/resveratrol fermentextract.

For purpose of gene microarray analysis, the term “up-regulation” or“up-regulated” implies that the gene is over-expressing RNA. The term“down-regulation” or “down-regulated” implies RNA is beingunder-expressed.

EXAMPLE—4 Full Thickness Evaluation

The yeast/resveratrol ferment extracts of the invention were tested onthe MatTek full thickness skin tissue model. This skin model consistsof: 1) normal human-derived epidermal keratinocytes that have beencultured to form a multilayered, highly differentiated model of thehuman epidermis and, 2) human fibroblasts that have been seeded into acollagen matrix to form the dermis. Upon arrival, the tissues werestored at 4° C. until used. For use, the tissues were removed from theagarose-shipping tray and placed into a 6-well plate containing 4 ml ofassay medium and allowed to equilibrate overnight at 37±2° C. and 5±1%CO2. After the overnight equilibration, the media was replaced with 4 mlof fresh media and 50 μl of test material was then applied topically tothe tissues. The tissues were then incubated for 24 hours at 37±2° C.and 5±1% CO2.

COX Assay

A membrane was equilibrated in Tris Buffered Saline (TBS: 20 mM Tris, pH7.5, 150 mM NaCl) and assembled into a Bio-Dot microfiltrationapparatus. After assembly, 200 μl of TBS was added to each well used inthe Bio-Dot and the vacuum was applied to ensure that there was adequateflow through all of the wells. Next, each cell culture media sample (400μl) or protein homogenate sample (approximately 5 μG) was assigned awell in the apparatus and was applied to the appropriate well. Thesamples were filtered under low vacuum. TBS was added to wells notassigned a sample to ensure that the membrane did not dry out during theprocedure. At the end of the blotting procedure and additional 200 μlwas applied and filtered through each well. The membrane was thenremoved from the Bio-Dot apparatus, washed in TBS for 5-10 minutes andthen placed into blocking solution (Tris Buffered Saline [20 mM Tris, pH7.5, 150 mM NaCl, 1% non-fat milk powder) and allowed to incubate for atleast 1 hour at room temperature on a rocking platform. The results ofthe assay are shown in FIG. 5.

Type IV Collagen Assay

A series of type I C-peptide standards was prepared ranging from 0 ng/mlto 500 ng/ml. An ELISA microplate was prepared by removing any unneededstrips from the plate frame. In each well to be used 100 μl of sample orstandard was added and the plate was incubated for 1 hour at 37° C.After the incubation, the plate was washed three times with wash buffer.After the last wash was removed, 100 μl of detection solution was addedto each well and the plate was incubated for 1 hour at 37° C. After theincubation, the plate was washed again as described above. When the lastwash was removed 100 μl of substrate solution was added to each well andthe plate was incubated at 37° C. for 30 minutes. After the incubation,50 μl of stop solution was added to each well and the plate was read at460 nm using a plate reader. The results of the assay are shown in FIG.6.

Melanin Assay on SkinEthic Tissue

SkinEthic's Tanned Epidermis model consists of normal, human-derivedepidermal keratinocytes and melanocytes that have been co-cultured toform a multilayered, highly differentiated model of the human epidermis.The melanocytes within this model undergo melanogenesis, leading to abasal level of melanin accumulation within the tissues over time whichcan be influenced by test materials that can either increase (skindarkening agents) or decrease (skin whitening agents) melanin synthesis.The results shown in FIG. 7 demonstrates that yeast/resveratrol fermentextract of the invention minimized the expression of melanin.

EXAMPLE—5 Preparation of Liposomal Encapsulated Yeast/ResveratrolFerment Extract

Samples of ‘yeast/resveratrol ferment extract’ were incorporated intoliposome comprising phospholipid and lecithin layer obtained fromsoybeans. The lysate was slurried together with liposome using ahigh-pressure homogenizer obtained from Hydraulic EngineeringCorporation (Brea, Calif.). The milky white mixture contained the‘yeast/resveratrol ferment extract’ encapsulated with the liposomalcomponents.

EXAMPLE—6 Preparation of Maltodextrin-Encapsulated Yeast/ResveratrolFerment Extract

Samples of ‘yeast/resveratrol ferment extract’ were encapsulated inmaltodextrin and spray-dried to essentially provide an anhydrous powderof maltodextrin-encapsulated ‘yeast/resveratrol ferment extract’ usingthe methodologies in WO2003/068161. The reference is incorporated hereinin its entirety.

The following proposed examples illustrate skin care compositionsaccording to the present invention that can be prepared using the‘yeast/resveratrol ferment extract’ as prepared using example-1.

PROPOSED EXAMPLE—7 Water-in-Oil Emulsion

The example illustrates a high internal phase water-in-oil emulsionincorporating the ‘yeast/resveratrol ferment extract’ prepared asdisclosed in Example-1.

Ingredient Wt % 1,3-dimethyl-2-imidazolidione 0.2 Polyoxylene (2) oleylether1 (Oleth-2) 5.0 Disteardimonium Hectorite 0.5 MgSO4•7H2O 0.3Preservative² 0.01 Yeast/Resveratrol Ferment extract 10.0 Water To 100

PROPOSED EXAMPLE—8 Oil-in-Water Cream

The example illustrates an oil-in-water cream incorporating the‘yeast/resveratrol ferment extract’ prepared as disclosed in Example-1.

Ingredient Wt % Mineral Oil 4 1,3-dimethyl-2-imidazolidione 1 Ceteth-104 Cetyl Alcohol 4 Triethanolamine 0.75 Butylene Glycol 3 Xanthum Gum 0.3Methyl, Propyl, and Butyl Paraben 0.01 Yeast/Resveratrol Ferment extract10.0 Water To 100

PROPOSED EXAMPLE—9 Alcoholic Lotion

The example illustrates an alcoholic lotion incorporating the‘yeast/resveratrol ferment extract’ prepared as disclosed in Example-1.

Ingredient Wt % 1,3-dimethyl-2-imidazolidione 0.3 Ethyl Alcohol 40Yeast/Resveratrol Ferment Extract 10.0 Water To 100

PROPOSED EXAMPLE—10 Sub-Micron Emulsion Concentrate

The example illustrates a sub-micron emulsion concentrate that containsthe ‘yeast/resveratrol ferment extract’ prepared as disclosed inExample-1.

Ingredient Wt % Trimethylopropane Tricaprylate/Tricaprate 18.0 Glycerin8.0 Cetcaryl alcohol 2.0 Cetcareth 20 2.0 Glyceral stearate 2.0Butylated Hydroxytoluene 0.01 Yeast/Resveratrol Ferment Extract 10.0Water To 100

PROPOSED EXAMPLE—11

Ingredient Wt % Water 89 ‘yeast/resveratrol ferment extract’ 10.0Preservative 1

What is claimed is:
 1. A method of producing yeast resveratrol fermentextract comprising: growing the cells of a yeast of Pichia genus in agrowth media to late-logarithmic growth phase, wherein the carbon sourcein said media is exhausted; exposing said cells to a non-cytotoxic doseof resveratrol; rupturing a yeast cell wall; and extracting cytoplasmicand extra-cellular components of the exposed yeast cells from the growthmedia.
 2. The method of claim 1, wherein the yeast is Pichia pastoris.3. The method of claim 1, wherein the media does not include anyanimal-derived products.
 4. The method of claim 1, wherein theresveratrol is dispersed in an organic solvent at a concentration offrom about 0.001 gram/liter to 1.0 gram/liter.
 5. The method of claim 1,wherein the yeast resveratrol ferment extract comprises cellularcomponents consisting of cellular protein material, cellular nucleicmaterial, cellular cytoplasmic material and cell wall components.
 6. Themethod of claim 1, wherein the extracted cytoplasmic and extra-cellularcomponents of the exposed cells are relatively water-soluble.
 7. Themethod of claim 1, further comprising forming a personal carecomposition comprising the yeast-resveratrol ferment extract and apreservative, wherein the preservative is selected from the groupconsisting of acids, alcohols, glycols, parabens, quaternary-nitrogencontaining compounds, isothiazolinones, aldehyde-releasing compounds,halogenated compounds, and combinations thereof.
 8. The method of claim7, wherein the preservative is present at a concentration between 0.01%and 10% by weight based on the total weight of the personal carecomposition.
 9. The method of claim 7, wherein the yeast-resveratrolferment extract is encapsulated within a encapsulant to control therelease rate of the extract, wherein the encapsulant is selected fromthe group consisting of liposomes, niosomes, sub-micron emulsions,polymeric encapsulates, gels, creams, lotions, and combinations thereof.10. The method of claim 7, wherein the personal care composition furthercomprises a cosmetically acceptable vehicle.
 11. The method of claim 7,wherein the personal care composition further comprises at least oneingredient selected from the group consisting of water, surfactants,emulsifiers, conditioners, emollients, waxes, oils, polymers,thickeners, fixatives, colorants, humectants, moisturizers, stabilizers,diluents, solvents, fragrances, botanicals, nutraceuticals,cosmeceuticals, therapeutics, pharmaceuticals, antifungals,antimicrobials, steroidal hormones, antidandruff agents, anti-acnecomponents, sunscreens, preservatives, and combinations thereof.